Isolation, structure and processing of bioactive peptides

Abstract

Amino acid sequences deduced from DNA sequences do not prove in which forms proteins, peptides and their processed products are present in the tissues. The products of a single gene may give rise to variants of peptides because of post-transcriptional or post-translational processing. Structural characterization of the isolated peptides and their multiple molecular forms is therefore essential. The aims of this study are to isolate novel forms of bioactive peptides from tissue extracts and to further develop detection and analysis strategies to facilitate the task.

A high throughput method was established, that combines chromatography with microphysiometry and structural analysis, for isolation and characterization of bioactive compounds present in low amounts in tissue extracts. The protocol was designed for isolation of orphan receptor ligands and its value was confirmed by isolation of a ligand (IGF-I) from porcine intestine.

A method was developed for C-terminal sequence analysis based on mass spectrometry, and applicable to a variety of Lys/Cys-containing and Pro/Arg-rich peptides. A technique that combines enzymatic digestion with capillary electrophoretic analysis of the cleavage products was established for determination of C-terminal amidation in peptides.

Several novel variants of peptide hormones were isolated. A bioactive N-terminally elongated proform of secretin was discovered in porcine intestine, and its presence suggests two alternative processing pathways for this prohormone. A novel form of CCK, nonsulfated CCK-58, biologically active in a guinea pig gall bladder contraction assay, was isolated from porcine intestine. The structure-activity relationship in the series of CCK peptides shows that the N-terminal segment of this hormone is in part compensating for the lowered activity observed with nonsulfated shortened CCK forms. A C-terminally elongated form of PHI, with distinct biological properties, was purified and provides a novel sequence of the porcine VIP/PHI precursor protein. The isolation from porcine spleen of PR-39 in high amount indicates that the PR-peptide may have a function beyond its antibacterial activity. A form of NK-lysin, previously only detected at the DNA level in porcine bone marrow, was isolated form porcine spleen and constitutes a new variant.

The results provide new insight into the biosynthesis, processing and structure/function relationships of the hormones.